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Microarray
Microarray Microarrays are used to evaluate the expression of a given gene from a cellular system. The DNA of living organisms contain a variety of genes. These genes encode for a variety of functional RNA and proteins that are expressed constitutively or conditionally-under given circumstances. When these genes are expressed they transcribed into mRNA and then mRNA can be translated into a polypeptide or protein. Microarray technology can assess genes that are constitutively expressed or conditionally expressed in the cell using DNA and RNA. How do Microarrays work? There are two parts to a microarray the base or solid phase and the variable phase. The base is composed of either glass, nylon membrane, or silicon chips. There are thousands of different regions on the base, to which thousands of different genes are attached , in an orderly fashion often using a computerized machine. The order is key as it enables researchers to later identify which genes are are being expressed or not. The genes may be in the form of DNA, cDNA, or oligonucleotides. (Also it is important to realize that more than one of the same gene is bound to the base in a given spot). The variable phase contains information from a cell or cells, depending on the study performed. To be more specific, RNA is isolated from the cell. The RNA is then converted into multiple copies of DNA using Reverse Transcriptase and PCR. The DNA nucleic acids included in this reaction will be fluorescently labeled, so following PCR the DNA will be associated with a given color. (When doing comparative studies between two cell types, one cell's cDNA will be one color and the other cell's cDNA a different color). The cDNA is incubated with the base and cDNA complementary to the attached DNA will hybridize. The expression of different mRNA will be detected through visualization of the fluorophores bound to the known attached gene. This can be done using laser technology, a special microscope, a camera, and computerized method for computation. A Simplified Example Let's say that you were interested in looking at the expression genes X, Y, and Z in two cells; one normal and one cancerous. You were doing this because you wanted to determine whether these genes were uniquely expressed in either the normal cell or cancer cell. To test this you would attach genes X, Y, and Z to a base (glass, silicon, or nylon) in a known order. Next you would separately isolate the mRNA of normal cells and mRNA of cancerous cells. (Remembering that expressed genes that transcribe DNA into mRNA). The mRNAs are separately subjected to reverse transcriptase and PCR- making multiple copies of cDNA. During this process the nucleotides associated with the normal cells are modified with green fluorophores and the nucleotides associated with cancerous cells are modified with red fluorophores. The cDNA of both cells is combined and incubated with the base. cDNA will hybridize to corresponding DNA. When the assay is subjected to visualizing techniques (a laser, a special microscope, a camera, and computerized method for computation) Different colors will appear at different regions of the base. Since the base DNA was ordered, we know which X, Y, Z genes have bound to cDNA, and therefore which mRNA is expressed in a cell. Given normal cDNA was labeled with green we can see which of the X,Y, Z genes lit up as green and were therefore uniquely expressed by the normal cells. Given cancerous cells were red we can see which of the X,Y, Z genes lit up as red and were therefore uniquely expressed by the cancerous cells. Regions of yellow represent both cancerous and normal expression and black signifies that neither cell expressed that gene. The intensity of the color can be used to determine the degree of expression in the cell. Some Studies utilizing microarray technology *Disease Characterization and Classification (cancer, heart disease, mental illness, infectious diseases) *Drug Development *Therapy Development *Disease Progression Useful Databases *GEO- Gene Expression Omnibus; the NCBI's effort to create an online database compiling gene expression information *MGED- Microarray Gene Expression Database; an independent effort to provide a compository of standardized gene expression data . New Technology This technology is utilized in gene chips. There have also been adaptations to the base with bead array technology with uses tiny beads. (For more info reference GeneChip and the Genographic Project Wiki) While perhaps not necessarily new technology, there are also protein, antibody, and microRNA microarrays. Protein microarrays look at protein-protein interaction, antibody microarrays look at antibody interactions. http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html http://www.genome.gov/10000533